硫化氢对大鼠肾上腺髓质嗜铬细胞分泌儿茶酚胺的影响及其机制研究

发布时间：2018-06-04 16:54

  本文选题：H_2S + 儿茶酚胺　； 参考：《山东大学》2012年硕士论文

【摘要】：研究背景 硫化氢(H2S)是一种具有臭鸡蛋气味的有毒气体。近年的研究表明,在人体内,硫化氢(H2S)是一种重要的气体信号分子,其通过作用于不同细胞的离子通道和受体在哺乳动物组织中发挥着重要的生理的调节功能,并且具有治疗心血管、神经、生殖和消化等方面疾病的潜在价值。人体内的L-半胱氨酸,可在胱硫醚β-合成酶(ystathionine β-synthase, CBS)和胱硫醚γ-裂解酶(cystathionine γ-lyase, CSE)作用下合成硫化氢。据相关文献报道,大鼠肾上腺髓质嗜铬细胞(adrenal chromaffin cells, AMCs)含有CSE。肾上腺髓质AMCs通过释放儿茶酚胺(catecholamine, CA)在应激反应的调节中发挥了关键作用。据文献报道缺氧可以导致CSE-/-型乳鼠AMCs的儿茶酚胺分泌减少。目前,对于外源性H2S能否诱导AMCs释放CA及其离子机制尚无报道。在本研究中,我们使用安培法和全细胞膜片钳记录法探讨外源性硫化氢能否诱发AMCs释放CA及其离子机制。 材料和方法 实验选用健康雌性Wistar大鼠(200-250g),自由饮食。实验前麻醉Wistar大鼠,取出肾上腺置于D-Hanks液中,剪除皮质,将每个髓质均分成两个,置于0.5ml混合酶液中(胶原酶Ⅰ3mg/ml、透明质酸酶2.4mg/ml、DNase0.5mg/ml、BSA2.6mg/ml)37℃恒温孵育70分钟,吸出酶液加入lml D-Hanks液冲洗组织块,再用巴斯德管吹打组织块,显微镜下观察到细胞后停止吹打,将细胞固定于盖玻片(用PLL处理过)上然后加入DMEM培养基(含10%的FBS),37℃恒温CO2培养箱中培养24小时。将固定有细胞的盖玻片放入记录腔室中,并用细胞外液持续灌流,速度为lml/min。使用5微米的碳纤维电极检测儿茶酚胺的释放。全细胞记录法检测嗜铬细胞的电流变化。 所有数据均采用均数±标准误表示,采用单因素方差分析比较多组间的差异,t检验比较两组间的差异,以P0.05为显著性差异界值。 结果 标准细胞外液中加入外源性的硫氢化钠(H2S供体)可以诱发AMCs释放CA。实验中使用70mMKCl作为阳性对照,细胞外液作为阴性对照。780mV电压时可以观察到碳纤维电极氧化CA产生的尖峰,切换到OmV时尖峰消失。将标准细胞外液换成无钙的细胞外液,硫氢化钠不能诱发AMCs释放CA。综上所述,H2S可以诱导AMCs释放CA。 同时使用安培记录法和全细胞记录法检测AMCs状态,发现加入外源性的硫氢化钠可以诱发AMCs去极化,并且伴有CA的分泌。 硫氢化钠可以抑制钙激活的钾通道。将细胞外液切换成无钙溶液时,硫氢化钠对的抑制作用消失。 结论 H2S能够直接诱发AMCs释放CA并且抑制K (Ca)电流。这一结论表明,硫化氢可能是通过调节肾上腺髓质K (Ca)的电流导致CA的释放进而参与哺乳类动物机体的应激反应。
[Abstract]:Research background Hydrogen sulfide (H 2 S) is a toxic gas with the smell of rotten eggs. Recent studies have shown that hydrogen sulfide (H _ 2S) is an important gas signaling molecule in human body, which plays an important physiological regulatory function in mammalian tissues through ion channels and receptors acting on different cells. It also has potential value in the treatment of cardiovascular, neurological, reproductive and digestive diseases. Human L-cysteine can synthesize hydrogen sulfide under the action of cystathionine 尾 -synthase (CBS) and cystathionine 纬 lyase (CSE). According to related literature, adrenal chromaffin cells, AMCs) in rat adrenal medullary cells contains CSE. Adrenomedullary AMCs plays a key role in the regulation of stress by releasing catecholamine. Hypoxia has been reported to reduce catecholamine secretion in AMCs of CSE-r-type neonatal mice. At present, whether exogenous H 2S can induce the release of CA from AMCs and its ion mechanism have not been reported. In this study, amperometric method and whole-cell patch clamp recording were used to investigate whether exogenous hydrogen sulfide could induce the release of CA from AMCs and its ion mechanism. Materials and methods Healthy female Wistar rats were fed a free diet of 200-250 g / g. Before the experiment, Wistar rats were anesthetized, the adrenal gland was taken out and placed in D-Hanks solution, the cortex was cut off, and each medulla was divided into two parts. Each medulla was incubated in 0.5ml mixed enzyme solution (collagenase 鈪，

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